【作者】 李飞；

【导师】 陆伦根；

【作者基本信息】 上海交通大学 ， 内科学（消化科）， 2017， 博士

【摘要】 研究目的:探索Y-box结合蛋白1(YB-1)在肝祖细胞(HPC)介导的肝再生和肝纤维化中的作用及其机制。研究方法:采用免疫荧光和免疫组化技术检测相关蛋白标志物在细胞或组织样本中的定性和定位表达。用慢病毒载体系统干扰HPC中YB-1的表达后,采用细胞计数试剂盒和流式细胞术检测HPC增殖和细胞周期。通过RNA-sequence分析沉默YB-1对HPC基因表达谱的改变,进一步阐明YB-1对HPC生物学功能的影响。采用染色质免疫共沉淀结合高通量测序技术(Ch IP-sequence)检测YB-1在HPC中的结合位点,并通过Ch IP-PCR进行验证。采用荧光素酶报告基因检测法探索YB-1对靶基因启动子转录活性的调控作用。RT-PCR和Western blot用以检测相关目的m RNA和蛋白的表达情况。研究结果:1正常人肝脏不表达YB-1,慢性乙型肝炎患者肝脏高表达YB-1,其中又以胆管细胞和肝祖细胞升高最为明显,并与肝纤维化程度呈正相关;2 Ch IP-sequence和RNA-sequence均提示YB-1能调控Hedgehog信号通路;Ch IP-sequence和Ch IP-PCR表明YB-1能与Gli2启动子区结合;荧光素酶报告基因实验表明YB-1能调控Gli2的转录;3沉默YB-1能抑制Gli2的表达,通过阻断Hedgehog信号通路而抑制HPC增殖;4 Ch IP-sequence和RNA-sequence提示YB-1能调控细胞粘附和细胞外基质相互作用;RT-PCR、Western blot和免疫荧光均表明肝祖细胞能分泌细胞外基质;5 Ch IP-sequence和Ch IP-PCR表明YB-1能与PDGFR-β启动子区结合并负调控其转录;荧光素酶报告基因实验表明YB-1能负调控PDGFR-β的转录;6沉默YB-1能促进HPC表达细胞外基质,并且PDGFR-β/ERK/p90RSK信号通路参与该过程。主要结论:YB-1能通过Hedgehog信号通路调控肝祖细胞增殖,通过PDGFR-β/ERK/p90RSK信号通路负调控肝祖细胞分泌细胞外基质。更多还原

【Abstract】 Objective: to explore the roles of Y-box protein-1(YB-1)in hepatic progenitor cells(HPC)and the detailed molecular mechanisms when liver regeneration and fibrogenesis develops.Methods : Immunofluorescence assays and immunohistochemistry technology were used to detect the protein expression in specific cells.After the expression of YB-1 in HPC,cell counting kit and flow cytometry were utilized to detect cell proliferation and cell cycles.RT-PCR and Western blot assays were performed to determine the m RNA and protein expression.Chromatin immunoprecipitation assays were introduced to determine the target genes of YB-1.Luciferase reporter assays were conducted to assess the regulatory effect of YB-1 on its target gene.Results(1)The expression of YB-1is elevated in livers of patients infected with chronic viral hepatitis B and the expression level correlated with the severity of liver fibrosis.(2)YB-1 directly binds to the promoter of Gli2 and regulate its transcriptional activity.YB-1 knockdown suppress the proliferation of HPC through down-regulated Hedgehog signaling.(3)YB-1 knockdown promotes the expression of extracellular matrix in HPC.PDGFR-β is a target gene of YB-1and YB-1 negatively regulates PDGFR-β promoter activity in HPCs.(4)PDGFR-β can regulate the expression of collagen I by ERK/p90 RSK signaling and disruption of the signaling pathway with PDGFR-β inhibitor or ERK1/2 inhibitor abolished the regulatory effect of PDGFR-β on collagen I expression in HPC.Conclusion: YB-1 can promote the proliferation of HPC via Hedgehog signaling and negatively regulate the expression of extracellular matrix through PDGFR-β /ERK/p90 RSK signaling.更多还原